PROJECT SUMMARY: Whole genome sequencing has revealed that mutations in regulatory proteins often account for the hypervirulent phenotype observed in bacteria causing severe infections. The major human pathogen group A Streptococcus (GAS) is well known to develop hypervirulence following mouse passage and during infection, but there is significant inter-strain variation in the rates of hypervirulence development. The main mutations driving GAS hypervirulence occur in the control of virulence sensor kinase, CovS, which represses virulence factor encoding genes via phosphorylation of its cognate response regulator, CovR. The GAS hyaluronic acid capsule has been considered essential to the hypervirulence enabled by CovS inactivation, but we have isolated two capsule- deficient serotype M4 GAS strains from a point-source mini-outbreak of invasive human infections. Despite being genetically identical, one of these isolates spontaneously acquires a CovS-inactivating mutation identical to that previously identified in a hypervirulent clinical serotype M1 strain. These findings set the stage for this proposal, which seeks to understand what intrinsic factors control the variation in the emergence of hypervirulence in GAS strains. In aim 1, we will assess whether acapsular GAS strains can develop hypervirulence via CovS inactivation by comparing the virulence of wild-type and CovS-inactivated acapsular serotype M4 strains using two murine models that mimic human disease. Additionally, the ability of acapsular serotype M4 strains to develop the CovS- inactivated, hypervirulent phenotype following mouse passage will be compared to that of an encapsulated serotype M1 strain. We have also identified key GAS cell surface proteins that are upregulated following CovS inactivation in an acapsular serotype M4 strain, but not in encapsulated M1 and M3 strains. We will analyze the role of these cell surface proteins in GAS hypervirulence by comparing serotype M4 strains that are wild-type, covS-inactivated and lacking specific CovRS-regulated downstream target genes, in a murine model of invasive GAS disease. Specific aim 2 will address the novel hypothesis that differential methylation patterns, and not DNA sequence, contribute to the emergence of hypervirulent GAS using our unique set of clinical serotype M4 strains that are genetically identical, but show differential ability to acquire CovS mutation. We will generate the first methylome data for hemolytic streptococci and correlate this with transcriptome data to identify the impact of methylation differences on gene regulation pathways that might influence the emergence of hypervirulence. Targeted inactivation of the GAS orphan DNA methylase will enable us to assess the contribution of these enzymes to observed methylation profiles. Completion of this research will bring us a step closer to answering the 100 year old question ?why only certain GAS strains develop hypervirulence? and help design more in-depth investigations designed to dissect the mechanisms underlying the emergence of hypervirulence in GAS.